C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity

Adrián Rafael Murillo-de-Ozores, Alejandro Rodríguez-Gama, Silvana Bazúa-Valenti, Karla Leyva-Ríos, Norma Vázquez, Diana Pacheco-Álvarez, Inti A. De La Rosa-Velázquez, Agnieszka Wengi, Kathryn L. Stone, Junhui Zhang, Johannes Loffing, Richard P. Lifton, Chao-Ling Yang, David Ellison, Gerardo Gamba, Maria Castañeda-Bueno

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overac-tivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4/ mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4’s C terminus at several positions increased its kinase activity toward Ste20-related proline/ alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-bind-ing site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Original languageEnglish (US)
Pages (from-to)12209-12221
Number of pages13
JournalJournal of Biological Chemistry
Volume293
Issue number31
DOIs
StatePublished - Jan 1 2018

Fingerprint

Protein Phosphatase 1
Phosphotransferases
Kidney
Binding Sites
Member 3 Solute Carrier Family 12
Proteolysis
Pseudohypoaldosteronism
Electrophoretic mobility
Metalloproteases
Protein Kinases
HEK293 Cells
Lysine
Salts
Genes
Tissue
Western Blotting
PAS domain kinases
Antibodies
Mutation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Murillo-de-Ozores, A. R., Rodríguez-Gama, A., Bazúa-Valenti, S., Leyva-Ríos, K., Vázquez, N., Pacheco-Álvarez, D., ... Castañeda-Bueno, M. (2018). C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity. Journal of Biological Chemistry, 293(31), 12209-12221. https://doi.org/10.1074/jbc.RA118.003037

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity. / Murillo-de-Ozores, Adrián Rafael; Rodríguez-Gama, Alejandro; Bazúa-Valenti, Silvana; Leyva-Ríos, Karla; Vázquez, Norma; Pacheco-Álvarez, Diana; De La Rosa-Velázquez, Inti A.; Wengi, Agnieszka; Stone, Kathryn L.; Zhang, Junhui; Loffing, Johannes; Lifton, Richard P.; Yang, Chao-Ling; Ellison, David; Gamba, Gerardo; Castañeda-Bueno, Maria.

In: Journal of Biological Chemistry, Vol. 293, No. 31, 01.01.2018, p. 12209-12221.

Research output: Contribution to journalArticle

Murillo-de-Ozores, AR, Rodríguez-Gama, A, Bazúa-Valenti, S, Leyva-Ríos, K, Vázquez, N, Pacheco-Álvarez, D, De La Rosa-Velázquez, IA, Wengi, A, Stone, KL, Zhang, J, Loffing, J, Lifton, RP, Yang, C-L, Ellison, D, Gamba, G & Castañeda-Bueno, M 2018, 'C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity', Journal of Biological Chemistry, vol. 293, no. 31, pp. 12209-12221. https://doi.org/10.1074/jbc.RA118.003037
Murillo-de-Ozores AR, Rodríguez-Gama A, Bazúa-Valenti S, Leyva-Ríos K, Vázquez N, Pacheco-Álvarez D et al. C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity. Journal of Biological Chemistry. 2018 Jan 1;293(31):12209-12221. https://doi.org/10.1074/jbc.RA118.003037
Murillo-de-Ozores, Adrián Rafael ; Rodríguez-Gama, Alejandro ; Bazúa-Valenti, Silvana ; Leyva-Ríos, Karla ; Vázquez, Norma ; Pacheco-Álvarez, Diana ; De La Rosa-Velázquez, Inti A. ; Wengi, Agnieszka ; Stone, Kathryn L. ; Zhang, Junhui ; Loffing, Johannes ; Lifton, Richard P. ; Yang, Chao-Ling ; Ellison, David ; Gamba, Gerardo ; Castañeda-Bueno, Maria. / C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity. In: Journal of Biological Chemistry. 2018 ; Vol. 293, No. 31. pp. 12209-12221.
@article{1d9ec07ac6314ef78d86aaf268ba6f11,
title = "C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity",
abstract = "WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overac-tivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4/ mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4’s C terminus at several positions increased its kinase activity toward Ste20-related proline/ alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-bind-ing site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.",
author = "Murillo-de-Ozores, {Adri{\'a}n Rafael} and Alejandro Rodr{\'i}guez-Gama and Silvana Baz{\'u}a-Valenti and Karla Leyva-R{\'i}os and Norma V{\'a}zquez and Diana Pacheco-{\'A}lvarez and {De La Rosa-Vel{\'a}zquez}, {Inti A.} and Agnieszka Wengi and Stone, {Kathryn L.} and Junhui Zhang and Johannes Loffing and Lifton, {Richard P.} and Chao-Ling Yang and David Ellison and Gerardo Gamba and Maria Casta{\~n}eda-Bueno",
year = "2018",
month = "1",
day = "1",
doi = "10.1074/jbc.RA118.003037",
language = "English (US)",
volume = "293",
pages = "12209--12221",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "31",

}

TY - JOUR

T1 - C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity

AU - Murillo-de-Ozores, Adrián Rafael

AU - Rodríguez-Gama, Alejandro

AU - Bazúa-Valenti, Silvana

AU - Leyva-Ríos, Karla

AU - Vázquez, Norma

AU - Pacheco-Álvarez, Diana

AU - De La Rosa-Velázquez, Inti A.

AU - Wengi, Agnieszka

AU - Stone, Kathryn L.

AU - Zhang, Junhui

AU - Loffing, Johannes

AU - Lifton, Richard P.

AU - Yang, Chao-Ling

AU - Ellison, David

AU - Gamba, Gerardo

AU - Castañeda-Bueno, Maria

PY - 2018/1/1

Y1 - 2018/1/1

N2 - WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overac-tivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4/ mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4’s C terminus at several positions increased its kinase activity toward Ste20-related proline/ alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-bind-ing site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

AB - WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overac-tivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4/ mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4’s C terminus at several positions increased its kinase activity toward Ste20-related proline/ alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-bind-ing site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

UR - http://www.scopus.com/inward/record.url?scp=85051119240&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85051119240&partnerID=8YFLogxK

U2 - 10.1074/jbc.RA118.003037

DO - 10.1074/jbc.RA118.003037

M3 - Article

VL - 293

SP - 12209

EP - 12221

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 31

ER -